RNase E is a 5′-end-dependent single strand endoribonuclease (Cormack and Mackie, 1992; Ehretsmann et al., 1992; Lin-Chao et al., 1994) that plays a general role in RNA metabolism and is a principal endoribonuclease in messenger RNA decay (Coburn and Mackie, 1999; Grunberg-Manago, 1999; Regnier and Arraiano, 2000). RNase E is a large, 1061 residue protein (Casaregola et al., 1992). Proteins related to RNase E are found throughout the eubacterial kingdom and in some plants (Condon et al., 2001). The plant homologues are presumably in the chloroplast, which is an organelle of eubacterial origin.
RNase E is believed to be responsible for degrading mRNA and processing rRNA and tRNA (Ow and Kushner, Genes and Development (2002)16:1102-1115). The mRNA degrading activity of RNase E significantly affects the efficiency of E. coli based protein expression systems. Bacteriophage T7 RNA polymerase (RNAP) elongates single strand RNA significantly faster than the E. coli enzyme. When mRNA is transcribed by T7 RNAP, long stretches of ribosome-free message occur. These untranslated mRNAs are very unstable, and their instability correlates with the rate of elongation of T7 PNAP (Makarova et al., Proc. Natl. Acad. Sci. USA 1995, 92, 12250-12254. RNase E is responsible for this rapid functional inactivation.
Furthermore, RNase E is responsible for processing the 9S precursors of the 5S rRNA and for processing tRNA. If the tRNA processing abilities of RNase E are disrupted, decreases in cell growth are observed and may be linked to the fact that RNase E cleavage is the rate-limiting step in the maturation of tRNAs.
The literature describes truncations of RNase E that possess limited ability to degrade mRNA, but maintain, at least in part, the ability to process rRNA and tRNA. The most extreme of these truncations, however, were created on plasmids. RNase E truncations created on plasmids, although relatively simple to construct, have disadvantages including the need to work in a recombination deficient background and the concern that the dose of the complementing gene could vary due to changes in plasmid copy number. Due to these limitations, RNase E mutations on plasmids are not suitable for use in exogenous protein production systems. A need, therefore, exists for a bacterial strain having a chromosomally integrated RNase E mutant that may be utilized to facilitate high levels of exogenous protein production.